The enhanced entry of extracellular Ca2+ is a major component of cellular Ca2+ signals generated by a variety of hormones and neurotransmitters acting on receptors coupled to phospholipase C (PLC). The focus of our research is to understand the nature of this Ca2+ entry, and its roles in overall intracellular signaling mechanisms. Interest in this field has largely focused on the so-called store- operated Ca2+ entry mediated by the Ca2+ release-activated Ca2+ (CRAC) channels whose gating is entirely dependent on, and subsequent to, the depletion of intracellular Ca2+ stores. More recently however, other store-independent pathways have been shown to play a key role, particularly at the lower, more physiologically relevant, levels of stimulation. Of these, the arachidonic acid-regulated Ca2+-selective (ARC) channels, that we first described some 14 years ago, remain the most thoroughly characterized. It is now known that both the CRAC and ARC channels are formed by members of the Orai protein family, with the CRAC channels being formed by Orai1 subunits, whilst a combination of Orai1 and Orai3 subunits form the ARC channels. A key protein in the activation of the CRAC channels is STIM1 which acts as the sensor of Ca2+ depletion of the ER stores, which initiates the translocation of STIM1 within the ER membrane to sites close the PM where it interacts with the CRAC channels to induce activation. Surprisingly, we found that STIM1 is also an essential component of ARC channel activation, but here is it the relatively minor pool of STIM1 that constitutive resides in the PM that is responsible and, in this case, the STIM1 is constitutively associated with the channel even under resting conditions. Critically, we have now shown that the two pools of cellular STIM1 (PM- and ER- resident) are not only spatially discrete, but also functionally discrete, with the result that these two co-existing, and biophysically very similar, conductance are capable of inducing entirely separate physiological responses with distinct spatial and temporal features. Importantly, current interest within the pharmaceutical industry to develop agents that target CRAC channel activities (e.g. in immunological and allergic responses), have focused on either Orai1 or STIM1 as potential targets. However, as is now clear, the involvement of both these proteins in the functionally distinct ARC channels means that such an approach will not be specific for the CRAC channel activities. In the proposed studies we will utilize a range of novel molecular tools and approaches that we have developed to examine the basis for the distinct mode of STIM1-dependent activation of the ARC channels, the physiological implications of this unique mode of activation and how it is modulated, and the specific roles of the ARC channels in physiologically-relevant cellular responses, particularly in relation to exocrine secretory activities. Delineating such unique features will not only help in revealing the range of unique physiological roles of the ARC channels but, ultimately, will also aid the development of a more refined approach to therapeutically targeting these two discrete pathways for agonist-induced cellular Ca2+ signaling.